Acoustic determination of properties of reservoirs and of fluids contained therein

ABSTRACT

The invention provides devices and methods for acoustically determining the properties of the contents of one or more reservoirs in a plurality of reservoirs. Each reservoir is adapted to contain a fluid. An acoustic radiation generator can be positioned in acoustic coupling relationship to each of the reservoirs. Acoustic radiation generated by the acoustic radiation generator is transmitted through each reservoir to an analyzer. The analyzer is capable of analyzing a characteristic of the transmitted acoustic radiation and optionally correlating the characteristic to a property of the reservoirs&#39; contents. Properties that may be determined include volume, temperature, and composition. The invention is particularly suited to determining the properties of the contents of a plurality of reservoirs to allow for accuracy and control over the dispensing of fluids therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.10/310,638, filed Dec. 4, 2002 now U.S. Pat. No. 6,938,995, which is acontinuation-in-part of U.S. patent application Ser. No. 10/010,972,filed Dec. 4, 2001, abandoned. The disclosures of the aforementionedapplications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The invention relates generally to the use of acoustic energy todetermine a temperature-dependent property of a fluid in a reservoirand/or of the reservoir-material itself. The determined properties canbe stored and optionally applied at a later time to enhance accuracy,precision, and efficiency in dispensing fluids from the reservoirs, andparticularly, in dispensing fluids with the use of focused acousticenergy. Moreover, the invention provides the capability of analyzing thecontents of a reservoir at any temperature, e.g., without having to thawa frozen well plate in which each well contains a frozen fluid.

BACKGROUND

The discovery of novel and useful materials depends largely on thecapacity to make and characterize new compositions of matter. As aresult, recent research relating to novel materials that have usefulbiological, chemical, and/or physical properties has focused on thedevelopment and implementation of new methods and systems forsynthesizing and evaluating potentially useful chemical compounds. Inparticular, high-speed combinatorial methods have been developed toaddress the general need in the art for systematic, efficient, andeconomical material synthesis techniques, as well as for methods toanalyze and to screen novel materials for useful properties.

Generally, it is important to control the quality of the startingmaterials in any chemical synthesis process. Otherwise, the integrity ofthe process and the quality of the resulting product are compromised.Quality control of the starting materials is a particularly importantissue in combinatorial synthesis procedures. In such procedures, a largenumber of starting compounds may be dispensed in a predeterminedsequence from a compound library to synthesize, for example, a batch ofa drug. As a further example, such procedures may be used in peptidedrug discovery applications to synthesize a drug containing a specificpeptide sequence. Should any of the starting compounds contain anunacceptable level of a contaminant or exhibit an unacceptable degree ofdegradation, the synthesized compound may be rendered useless. Ineffect, all starting compounds employed for the batch synthesis would bewasted. This is particularly problematic when one or more of thestarting compounds are rare or expensive.

Similarly, combinatorial testing techniques may be employed inanalytical and testing procedures. For example, a combination of two ormore pharmacologically active candidate compounds may be delivered to atest sample in order to assess whether synergistic effects are achieved.If any one of the candidate compounds is compromised in quality,however, the accuracy and reliability of the assessment may be reduced.Thus, further testing may be necessary, adding significantly to theoverall time and cost associated with the combinatorial testing process.

High-speed combinatorial methods often involve the use of arraytechnologies that require accurate dispensing of fluids, each having aprecisely known chemical composition, concentration, stoichiometry,ratio of reagents, and/or volume. Such array technologies may beemployed to carry out various synthetic processes and evaluations. Arraytechnologies may use large numbers of different fluids to form aplurality of reservoirs that, when arranged appropriately, createcombinatorial libraries. In order to carry out combinatorial methods, anumber of fluid dispensing techniques have been explored, such as pinspotting, pipetting, inkjet printing, and acoustic ejection. Many ofthese techniques possess inherent drawbacks that must be addressed,however, before the fluid dispensing accuracy required for thecombinatorial methods can be achieved. For instance, a number of fluiddispensing systems are constructed using networks of tubing or otherfluid-transporting vessels. Tubing, in particular, can entrap airbubbles, and nozzles may become clogged by lodged particulates. As aresult, system failure may occur and cause spurious results.Furthermore, cross-contamination between the reservoirs of compoundlibraries may occur due to inadequate flushing of tubing and pipettetips between fluid transfer events. Cross-contamination can easily leadto inaccurate and misleading results.

Acoustic ejection provides a number of advantages over other fluiddispensing technologies. In contrast to inkjet devices, nozzleless fluidejection devices are not subject to clogging and their associateddisadvantages, e.g., misdirected fluid or improperly sized droplets.Furthermore, acoustic technology does not require the use of tubing orinvolve invasive mechanical actions, for example, those associated withthe introduction of a pipette tip into a reservoir of fluid.

Acoustic ejection has been described in a number of patents. Forexample, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquiddrop emitter that utilizes acoustic principles to eject droplets from abody of liquid onto a moving document to result in the formation ofcharacters or barcodes thereon. A nozzleless inkjet printing apparatusis used such that controlled drops of ink are propelled by an acousticalforce produced by a curved transducer at or below the surface of theink. Similarly, U.S. Pat. No. 6,666,541 to Ellson et al. describes adevice for acoustically ejecting a plurality of fluid droplets towarddiscrete sites on a substrate surface for deposition thereon. The deviceincludes an acoustic radiation generator that may be used to eject fluiddroplets from a reservoir, as well as to produce a detection acousticwave that is transmitted to the fluid surface of the reservoir to becomea reflected acoustic wave. Characteristics of the reflected acousticradiation may then be analyzed in order to assess the spatialrelationship between the acoustic radiation generator and the fluidsurface. Thus, acoustic ejection may provide an added advantage in thatthe proper use of acoustic radiation provides feedback relating to theprocess of acoustic ejection itself.

Regardless of the dispensing technique used, however, inventory andmaterials handling limitations generally dictate the capacity ofcombinatorial methods to synthesize and analyze increasing numbers ofsample materials. For instance, during the formatting and dispensingprocesses, microplates that contain a plurality of fluids in individualwells may be thawed, and the contents of selected wells can then beextracted for use in a combinatorial method. When a pipetting system isemployed during extraction, a minimum loading volume may be required forthe system to function properly. Similarly, other fluid dispensingsystems may also require a certain minimum reservoir volume to functionproperly. Thus, for any fluid dispensing system, it is important tomonitor the reservoir contents to ensure that at least a minimum amountof fluid is provided. Such content monitoring generally serves toindicate the overall performance of a fluid dispensing system, as wellas to maintain the integrity of the combinatorial methods.

During combinatorial synthesis or analysis processes, environmentaleffects may play a role in altering the contents of reservoirs on a wellplate or microplate. For example, dimethylsulfoxide (DMSO) is an organicsolvent commonly employed to dissolve or suspend many of the compoundsfound in drug libraries. DMSO is highly hygroscopic and tends to absorbambient water with which it comes into contact. In turn, the absorptionof water dilutes the concentration the compounds as well as alters theability of the DMSO to dissolve or suspend the compounds. Furthermore,the absorption of water may promote the decomposition of water-sensitivecompounds.

Acoustic monitoring makes it possible to determine the DMSO content ofDMSO/water mixtures in microplate reservoirs, for DMSO concentrationsranging from about 70 to 100% by volume. When the fluid in themicroplate well is a binary mixture of DMSO and water, adding water toor removing water from the mixture results in a change in the echoamplitude from the fluid/well-bottom interface. Measurement of theamplitude of reflection from microplate surfaces enables a calculationof acoustic impedance for the liquid in the microplate well. Whenacoustic impedance is translated to DMSO concentration in a DMSO/watermixture by the appropriate empirical model, the results are accurate towithin a few percentage points at room temperature.

However, if the microplate temperature deviates from room temperature,both the acoustic impedance of the microplate, and potentially that ofthe fluid in the wells, will change. As a consequence, the change in theamplitude of reflection for acoustic energy at the fluid/well interfacewill be interpreted as a change in DMSO/water composition.

For example, if a microplate containing a frozen DMSO/water mixture isprocessed by an acoustic instrument, e.g., the Echo 550 instrument(Labcyte Inc., Sunnyvale, Calif.), the initial DMSO content isincorrectly measured as being less than 40%. It takes nearly one hourbefore the microplate warms to room temperature, when the equilibriumacoustic impedances needed for the room temperature empirical modeldescribed above are reached, so that the composition of the DMSO/watermixtures in the wells can be accurately measured.

Benefits in accuracy and in time used would be obtained if microplatesdid not have to be at thermal equilibrium in order to accurately measurefluid composition in a well. A device and method is needed for makingaccurate compositional measurements without having to bring well platesto room temperature, by compensating for thermal variations in acousticimpedance and changes in the resulting echoes.

A number of patents describe the use of acoustic energy to assess thecontents of a container. U.S. Pat. No. 5,507,178 to Dam, for example,describes a sensor for determining the presence of a liquid and foridentifying the type of liquid in a container. The ultrasonic sensordetermines the presence of the liquid through an ultrasonic liquidpresence sensing means and identifies the type of liquid through aliquid type identification means, and includes a pair of electrodes andan electrical pulse generating means. This device suffers from thedisadvantage that the sensor must be placed in contact with the liquid.

U.S. Pat. No. 5,880,364 to Dam, on the other hand, describes anon-contact ultrasonic system for measuring the volume of liquid in aplurality of containers. An ultrasonic sensor is disposed opposite tothe tops of the containers. A narrow beam of ultrasonic radiation istransmitted from the sensor to the open top of an opposing container,and is then reflected from the air-liquid interface of the containerback to the sensor. By using the round trip transit time of theradiation and the dimensions of the containers being measured, thevolume of liquid in the container can be calculated. This device cannotbe used to assess the contents of sealed containers. In addition, thedevice lacks precision because air is a poor conductor of acousticenergy. Thus, while this device may provide rough estimates of thevolumes of liquid in relatively large containers, it is unsuitable foruse in providing a detailed assessment of the contents of reservoirstypically used in combinatorial techniques. In particular, this devicecannot determine the position of the bottom of containers, sincesubstantially all of the emitted acoustic energy is reflected from theliquid surface and does not penetrate to detect the bottom. Low volumereservoirs in microplates are regular arrays of fluid containers, andthe location of the bottoms of the containers can vary by a significantfraction of the nominal height of a container due to distortions in theplate, such as bowing. Thus, detection of only the position of theliquid surface leads to significant errors in height and thus volumeestimation in common containers.

There is thus a need in the art for improved methods and devices thatare capable of determining temperature-dependent properties ofreservoirs and fluids contained therein. Such capabilities would beparticularly useful for tracking the quality of compound libraries insolution, and the quantities of the compounds therein, for combinatorialand screening applications. In particular, it would be useful todetermine such properties for a range of reservoir and microplatetemperatures.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method for acousticallydetermining a temperature-dependent property of a fluid in a reservoir,the reservoir being integral with or located on a substrate. The methodcomprises the following steps:

(a) acoustically coupling an acoustic radiation generator and thereservoir, with the acoustic radiation generator located external to thereservoir;

(b) activating the acoustic radiation generator to generate acousticradiation and direct the acoustic radiation generated into and throughthe substrate toward the reservoir, such that the acoustic radiation istransmitted into the fluid in the reservoir and thereafter reflectedfrom an interior surface of the reservoir;

(c) measuring a temperature-dependent characteristic of the reflectedacoustic radiation, said characteristic selected so as to correlate witha temperature-dependent property of the fluid; and

(d) analyzing the characteristic to determine the temperature-dependentproperty of the fluid.

In a preferred embodiment of the present invention, the property of thefluid to be analyzed is fluid composition, e.g., the ratio of one fluidto another in any given reservoir. An example of such an analysisinvolves determining the ratio of DMSO to water in a mixture of the twosolvents, as DMSO/water mixtures are often used to dissolve or suspendcompounds commonly found in drug libraries.

Typically, well plates and microplates that contain a plurality ofreservoirs are stored prior to use under refrigerated conditions, tochill or freeze the fluids in the reservoirs prior to use. In the methodof the present invention, it is possible to provide information to theanalyzer to determine the temperature of frozen fluids in a well plateand thereby to process acoustic radiation reflected at a frozenfluid/reservoir interface to determine the composition of such frozenfluid. The method may also be used to measure the temperature of thewell plate.

In another embodiment, then, a method is provided for acousticallydetermining a temperature-dependent property of a reservoir material,the method comprising:

(a) acoustically coupling an acoustic radiation generator and thereservoir, with the acoustic radiation generator located external to thereservoir;

(b) activating the acoustic radiation generator to generate acousticradiation and direct the acoustic radiation generated through anexterior surface of the reservoir toward an interior surface of thereservoir such that acoustic radiation is thereafter reflected from aninterior surface of the reservoir;

(c) measuring a temperature-dependent characteristic of the reflectedacoustic radiation, said characteristic selected so as to correlate witha temperature-dependent property of the reservoir material; and

(d) analyzing the characteristic to determine the temperature-dependentproperty of the reservoir material.

In still another embodiment, the invention relates to a method foracoustically determining a property of a fluid disposed in a reservoiror, optionally, in each of a plurality of fluid reservoirs. The methodinvolves selecting a reservoir from the plurality of reservoirs, eachcomprising a solid surface disposed on a substrate, wherein a portion ofeach reservoir is adapted to contain a fluid, and positioning anacoustic radiation generator in an acoustically coupled relationship tothe selected reservoir. Once positioned, the acoustic radiationgenerator is actuated so that generated acoustic radiation istransmitted through the selected reservoir to be reflected from aninterface between the fluid and the reservoir to an analyzer forprocessing. Even if the well is empty or the measurement is made from asidewall rather than from the bottom, acoustic radiation directedthrough the chosen site will be reflected from the air/site orfluid/site interface to the analyzer. The analyzer then processes thereflected radiation to determine a characteristic thereof, whichprovides information about a property of the fluid in the selectedreservoir. Optionally, the acoustic radiation generator can berepositioned to provide information about a property of the fluid in theremaining reservoirs.

Typically, a property of the fluid in the selected reservoir isdetermined by processing the acoustic radiation transmitted through thereservoir. The results of the acoustic analysis may be storedelectronically for later use.

Typically, the inventive device includes a single acoustic radiationgenerator and a plurality of removable reservoirs. However, theinventive device can comprise a plurality of acoustic generators, suchas those described in U.S. patent application Ser. No. 10/310,638 toMutz et al., which each span more than one well. In addition, theacoustic radiation generator may comprise a component common to theanalyzer, such as a piezoelectric element. Optionally, the acousticgenerator may represent a component of an acoustic ejector, which ejectsdroplets from the reservoirs. In such a case, the device may furthercomprise a means to focus the acoustic radiation.

In a further embodiment, the invention provides a device foracoustically determining a property of a fluid in a reservoir adapted tocontain the fluid, the reservoir being contained in or disposed on asolid surface of a substrate. The device includes an acoustic radiationgenerator for generating acoustic radiation. The device also includesmeans for positioning the acoustic radiation generator in anacoustically coupled relationship to the reservoir. The acousticradiation generator is then actuated to transmit acoustic radiationthrough the reservoir, to be reflected from an interface between thefluid and the reservoir. The acoustic radiation reflected from theinterface is delivered to an analyzer for processing. The analyzer thenprocesses the reflected acoustic radiation to determine a characteristicthereof that provides information about a property of the fluid in thereservoir. Even if the reservoir is empty, the reflected acoustic energyfrom the air/reservoir interface can still be used to compute thetemperature of the well plate material at the reservoir.

Reflections from other interfaces in the well plate can be subjected toa similar analysis, e.g., the reflection from the bottom of themicroplate and from the top of the microplate wall in between wells.Transit times for such reflections can be used, for example, todetermine temperatures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B, collectively referred to as FIG. 1, schematicallyillustrate in simplified cross-sectional view a preferred embodiment ofthe inventive device. This embodiment allows both the acousticassessment, in reflective mode, of the contents of a plurality ofreservoirs, and the ejection of fluid droplets therefrom. As depicted,the device comprises first and second reservoirs, a combined acousticanalyzer and ejector, and an ejector positioning means. FIG. 1A showsthe acoustic ejector acoustically coupled to the first reservoir; theejector is activated in order to eject a droplet of fluid from withinthe first reservoir toward a site on a substrate surface to form afeature of an array. FIG. 1B shows the acoustic ejector acousticallycoupled to a second reservoir.

FIG. 2 schematically illustrates in simplified cross-sectional view anembodiment of the inventive device designed to permit acousticalassessment of the contents of a plurality of reservoirs in transmissivemode.

FIG. 3 is a chart showing measurements of apparent DMSO concentration ina sealed microplate as it was warmed to room temperature, calculatedwithout correcting for the temperature.

FIG. 4 A-C schematically displays the use of the travel time of anacoustic wave in a well plate to determine temperature, and FIG. 4D is atop view of a well.

FIG. 5 displays a model for use in the analyzer to determine theacoustic impedance change with temperature for both the microplate andthe fluid in the reservoir or well.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific fluids,biomolecules, or device structures, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a reservoir” includes a plurality of reservoirs, referenceto “a fluid” includes a plurality of fluids, reference to “abiomolecule” includes a combination of biomolecules, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set forthbelow.

When a first object is stated to be “acoustically coupled to” or “in anacoustic coupling relationship with” a second object, it is intendedthat the objects are positioned so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two entities are indirectly acoustically coupled, an“acoustic coupling medium” provides an intermediate medium through whichacoustic radiation may be transmitted. For example, an acoustic ejectormay be acoustically coupled to a fluid by immersing the ejector in thefluid or by interposing an acoustic coupling medium between the ejectorand the fluid, in order to transfer acoustic radiation generated by theejector through the acoustic coupling medium and into the fluid.

The terms “biomolecule” and “biological molecule” are usedinterchangeably herein to refer to any organic molecule that is, was, orcan be a part of a living organism, regardless of whether the moleculeis naturally occurring, recombinantly produced, or chemicallysynthesized in whole or in part. The terms encompass, for example,nucleotides, amino acids, and monosaccharides, as well as oligomeric,polymeric, and macromolecular species, such as oligonucleotides,polynucleotides, polypeptides, proteins, disaccharides,oligosaccharides, polysaccharides, mucopolysaccharides, peptidoglycans,and the like.

The term “fluid,” as used herein to refer to the material that the“reservoirs” herein are adapted to contain, generally although notnecessarily refers to matter that is at least partially nonsolid, or atleast partially gaseous and/or liquid, but not entirely gaseous. A“partially nonsolid fluid” will generally be a mixture of a liquid and asolid that is minimally, partially, or fully solvated, dispersed, orsuspended in the liquid. Examples of fluids include, without limitation,aqueous liquids, including water per se and salt water, and nonaqueousliquids such as organic solvents and the like. Combinations of fluidsare encompassed by the term as well, and include, by way of example,compositions containing two or more miscible and/or immiscible liquids.For the purpose of the present invention, a fluid in a reservoir may becooled to a temperature at which the fluid becomes partially orcompletely frozen, and will sometimes still be referred to herein as a“fluid.”

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point, either bya device separate from the acoustic energy source that acts like anoptical lens, or by the spatial arrangement of acoustic energy sourcesto effect convergence of acoustic energy at a focal point byconstructive and destructive interference. A focusing means may be assimple as a solid member having a curved surface, or it may includecomplex structures such as those found in Fresnel lenses, which employdiffraction in order to direct acoustic radiation. Suitable focusingmeans also include phased array methods as are known in the art anddescribed, for example, in U.S. Pat. No. 5,798,779 to Nakayasu et al.and Amemiya et al. (1997) Proceedings of the 1997 IS&T NIP13International Conference on Digital Printing Technologies, pp. 698-702.

The term “moiety” refers to any composition of matter or a fragment ormixture thereof, e.g., e.g., a molecular fragment, an intact molecule(including a monomeric molecule, an oligomeric molecule, and a polymer),or a mixture of materials (for example, an alloy or a laminate).

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

The term “reservoir” as used herein refers to any means for containing afluid, e.g., a receptacle or chamber. In some instances the reservoir iscontained in or disposed on a solid surface of a substrate. A fluidcontained in a reservoir necessarily will have a free surface, e.g., asurface that allows acoustic radiation to be reflected therefrom or asurface from which a droplet may be acoustically ejected. A reservoirmay also be a locus on a substrate surface within which a fluid can beconstrained. A reservoir may also be a well in a well plate.

The term “substrate” as used herein refers to any material that (1) hasa surface onto which one or more fluids may be deposited, (2) containsone or more fluid reservoirs, or (3) can serve as a support for one ormore fluid reservoirs permanently affixed thereto or otherwise placedthereon.

The invention accordingly relates to methods and devices foracoustically determining one or more properties of the contents of aplurality of fluid reservoirs.

The device used in conjunction with the present method generally,although not necessarily, includes a plurality of reservoirs, eachadapted to contain a fluid, and an acoustic radiation generator forgenerating acoustic radiation. The device also includes a means foracoustically coupling the acoustic radiation generator with a reservoirand/or a fluid contained therein, such that acoustic radiation generatedby the acoustic radiation generator is transmitted through at least aportion of the reservoir. An analyzer for analyzing atemperature-dependent characteristic of acoustic radiation reflectedfrom an interior surface of the reservoirs is positioned to receive thereflected acoustic radiation.

The device may include the reservoirs as integral or permanently affixedcomponents of the device. However, to provide for modularity andinterchangeability of components, it is preferred that the reservoirs tobe removable from the device. Generally, the reservoirs are arranged ina pattern or an array to provide each reservoir with individualsystematic addressability. In addition, while discrete reservoirs may beprovided, in circumstances that require a large number of reservoirs, itis preferred that the reservoirs are attached to each other or representintegrated portions of a single reservoir unit. For example, thereservoirs may represent individual wells in a well plate. Many wellplates suitable for use with the device are commercially available andmay contain, for example, 96, 384, 1536, or 3456 wells per well plate.Manufacturers of suitable well plates for use in the employed deviceinclude Corning, Inc. (Corning, N.Y.) and Greiner America, Inc. (LakeMary, Fla.). However, the availability of such commercially availablewell plates does not preclude the manufacture and use of custom-madewell plates containing at least about 10,000 wells, or as many as100,000 to 500,000 wells, or more.

Furthermore, the material used in the construction of reservoirs must becompatible with the fluids contained therein. Thus, if it is intendedthat the reservoirs contain an organic solvent such as acetonitrile,polymers that dissolve or swell in acetonitrile would be unsuitable foruse in forming the reservoirs or well plates. Similarly, reservoirs orwells intended to contain DMSO must be compatible with DMSO. Forwater-based fluids, a number of materials are suitable for theconstruction of reservoirs and include, but are not limited to, ceramicssuch as silicon oxide and aluminum oxide, metals such as stainless steeland platinum, and polymers such as polyester andpolytetrafluoroethylene. For fluids that are photosensitive, thereservoirs may be constructed from an optically opaque material that hassufficient acoustic transparency for substantially unimpairedfunctioning of the device.

Generally, a single acoustic radiation generator is used, though aplurality of acoustic radiation generators may be used. All acousticradiation generators employ a vibrational element or transducer togenerate acoustic radiation. Commonly, a piezoelectric element isemployed to convert electrical energy into mechanical energy associatedwith acoustic radiation. When a single acoustic radiation generator isused, the positioning means should allow for the acoustic radiationgenerator to move from one reservoir to another quickly and in acontrolled manner, thereby allowing fast and controlled scanning of thecontents of the reservoirs.

In some instances, the analyzer is positioned in fixed alignment withrespect to the acoustic radiation generator. In other instances,however, a means similar to that described above is provided foraltering the relative position of the analyzer with respect to thereservoirs. The relative position of the analyzer and the acousticradiation generator depends on the particular configuration of thedevice. In some instances, the device may be configured to operate intransmissive mode, such that the generated radiation is transmittedthrough the entirety of the reservoir undergoing analysis. In such acase, the reservoir may be interposed between the acoustic radiationgenerator and an acoustic analyzer. As another option, the device may beconfigured to operate in a reflective mode, such that the acousticradiation is transmitted only through a portion the reservoir whosecontents are being assessed. In such a case, the analyzer may bepositioned in a manner appropriate for this configuration, e.g., inorder to receive reflected acoustic radiation. In any case, the acousticradiation generator should be positioned such that generated acousticradiation is transmitted through the portion of each reservoir mostlikely to contain a fluid, for optimal performance. This reduces thechance that the analyzer will erroneously determine that a reservoir isempty. For example, as fluids ordinarily flow to the bottom ofcontainers or are driven there by centrifugation, the acoustic radiationgenerator should be positioned such that generated acoustic radiation istransmitted through the bottom of a reservoir.

In a preferred configuration, as discussed in detail below, the analyzeris positioned to receive acoustic radiation reflected from a freesurface of a fluid contained in each reservoir. In such a configuration,the acoustic radiation generator may comprise a component common to theanalyzer. The component common to the acoustic radiation generator andthe analyzer may be a vibrational element that converts one form ofenergy into another, e.g., a piezoelectric element that convertsacoustic/mechanical energy to electrical energy.

As discussed above, the reservoirs may be constructed to reduce theamount of movement and time needed to align the acoustic radiationgenerator with each reservoir or reservoir well during operation. As ageneral matter of convenience and efficiency, it is desirable to analyzean entire library of different moieties in a relatively short amount oftime, e.g., about one minute. Thus, the method typically allows for theanalysis of a fluid in the reservoirs at a rate of at least about 96reservoirs per minute. Faster analysis rates of at least about 384,1536, and 3456 reservoirs per minute are achievable with present daytechnology as well. Thus, the method can be implemented to analyze theproperties of the fluid contents of each well of most (if not all) wellplates that are currently commercially available. Proper implementationof the inventive method should yield a reservoir analysis rate of atleast about 10,000 reservoirs per minute. Current commercially availablepositioning technology allows the acoustic radiation generator to bemoved from one reservoir to another, with repeatable and controlledacoustic coupling at each reservoir, in less than about 0.1 second forhigh performance positioning means and in less than about 1 second forordinary positioning means. A custom designed system will allow theacoustic radiation generator to be moved from one reservoir to anotherwith repeatable and controlled acoustic coupling in less than about0.001 second.

In addition, to reduce the amount of movement and time needed to alignthe acoustic radiation generator with each reservoir or reservoir wellduring operation, as discussed infra, it is preferable in some instancesthat the center of each reservoir be located not more than about 1centimeter, preferably not more than about 1 millimeter, and optimallynot more than about 0.5 millimeter, from a neighboring reservoir center.These dimensions tend to limit the size of the reservoirs to a maximumvolume. The reservoirs are constructed to contain typically no more thanabout 1 mL, preferably no more than about 1 μL, and optimally no morethan about 1 nL, of fluid. To facilitate handling of multiplereservoirs, it is also preferred that the reservoirs be substantiallyacoustically indistinguishable. In other embodiments, when working withsmall fluid volumes is unnecessary, the reservoirs may have a volume of1 mL or greater, although reservoir volume will generally tend to beless than 500 μL.

By analyzing acoustic radiation that has been transmitted through atleast a portion of a selected reservoir, one may accurately determine atemperature-dependent property of a fluid contained in a reservoirand/or of the reservoir material itself. As discussed above, fluidproperties that may be determined include, but are not limited to,viscosity, surface tension, temperature, acoustic impedance, solidcontent, and impurity content. In some instances, the assessment mayinvolve measuring the travel time of acoustic radiation through thereservoir. For example, the transit time for sound through a well platebottom can be used as a thermometer for the microplate at a given welllocation.

For most materials, the speed of sound changes with temperature. Bysampling sound reflected from the microplate material interfaces at ahigh enough rate, the change in the transit time due to the temperaturedependence of the speed of sound can be detected. For example samplingat a rate that is in excess of 100 times the transit time could detect a1% change in the speed of sound. Higher sampling rates would allow formore sensitive measurements and for the use of thinner materials.

Polypropylene is a material commonly used to make the microplatesemployed in compound library storage. The travel time through apolypropylene microplate bottom increases as it warms, due to thereduction in the speed of sound with temperature typical for apolypropylene material. For example, Profax™ Polypropylene shows a0.342% reduction in the speed of sound per ° C. at temperatures nearroom temperature (approximately 22° C.). If a Profax Polypropylene plateexperienced a 10% reduction in the speed of sound as it warmed to roomtemperature, an approximate 30° C. warming of the microplate bottomwould be indicated.

If the transit time is known for a plate that is made of a material forwhich the speed of sound is a known function of temperature, the platecan act as a thermometer. This transit time would give a “bulktemperature” based on the average velocity of sound through the wellbottom. If the “bulk temperature” were indicative of the fluid/plateinterface temperature, then accurate values of impedance for thematerials can be used to determine the reflected echo amplitude. Fromsuch data, a temperature-corrected/compensated impedance model and areflection amplitude curve can be constructed or empirically derived.

The reflected wave at an interface depends on the acoustic impedances(Z) of the two materials forming the interface, e.g., Zfld of the fluidand Zwp of the well plate. Because both Zfld and Zwp change withtemperature, the magnitude of the reflected wave used to determine thefluid composition also changes with temperature. Knowledge of thetemperature of already characterized materials at the interface providesimpedance information for the microplate, and enables the properinterpretation of the reflected amplitude as a function of the impedanceof the fluid. The impedance information combined with temperatureinformation provides an accurate measure of fluid composition. Accurateinformation about the fluid cannot be obtained without knowledge of thetemperature of the materials and of the impedance of the microplatematerial. Determining the material composition of fluids withinreservoirs, e.g., wells in microplates, involves an analysis of thereflection of acoustic energy from interfaces such as that between thefluid and the bottom, interior surface of the reservoir. The amount ofreflected acoustic energy, R, from the interface and the transmittedenergy, T, across the interface can be calculated from the acousticimpedance, Z, of the interface materials. For a microplate plastic withacoustic impedance of Z_(p) and a well fluid with acoustic impedance ofZ_(f) the reflected and transmitted acoustic energy is given byEquations (1a) and (1b):

$\begin{matrix}{R = \left( \frac{Z_{p} - Z_{f}}{Z_{p} + Z_{f}} \right)^{2}} & \left( {1a} \right) \\{T = \frac{4\; Z_{p}Z_{f}}{\left( {Z_{p} + Z_{r}} \right)^{2}}} & \left( \text{1b} \right)\end{matrix}$

The acoustic impedance changes with temperature for both the microplateand the well fluid (or solid at temperatures below the freezing point ofthe fluid) can be utilized over a broad temperature range (minus 80° C.up to a “warm” room temperature). The method of using the transit timein thermometry is a critical first step in the process of obtaining an“in situ” temperature measurement at the interface between the well andwell contents for each well in a microplate, so that the properimpedances based on temperature are obtained and used.

An improvement to the above temperature measurement method is to use thereflection amplitude from the microplate bottom at room temperature toconfirm that the acoustic impedance of the microplate material matchesthe expected value for a room temperature measurement. This surfaceshould be at the same temperature as the liquid used to provide anacoustic path to the well.

Note that the dependence of the speed of sound, reflection amplitude,and acoustic impedance on temperature can be approximated by linearfunctions over a certain range of temperatures. Deviations from thislinear behavior are seen in some materials used in acousticapplications, as well as in most materials over broad temperatureranges. In particular, temperature-dependent properties can exhibitanomalies at phase change boundaries, such as at the melting point ofthe fluid in the reservoir, when both liquid and solid material maycoexist in the reservoir.

In addition, or in the alternative, the assessment of reservoir contentsmay involve determining the difference of acoustic radiation propertiesbefore and after transmission through the reservoir. Fortemperature-dependent properties, temperature measurement means known inthe art, such as thermocouples, may be used in conjunction with suchanalyses. Or, as suggested above, for a known material and/or geometry,a temperature-dependent property such as the speed of sound or acousticimpedance can be determined from an acoustic measurement; this can beused to establish the temperature where the measurement was taken.

Optionally, the results of acoustic analysis performed by the acousticanalyzer may be stored. Thus, the inventive device may include, forexample, a storage means comprising rewritable and/or permanent datastorage media for storing the results of acoustic analysis performed bythe analyzer.

Acoustic assessment as described above may be employed to improve fluiddispensing from each of a plurality of reservoirs adapted to contain afluid. Thus, another embodiment of the invention relates to a device fordispensing fluid from each of a plurality of reservoirs adapted tocontain a fluid. This device may include any of a number of knowntechniques for dispensing fluids involving contact-based fluiddispensing, e.g., pin spotting, pipetting, and inkjet printing, ornon-contact based fluid dispensing, e.g., acoustic ejection. However,the device represents an improvement over the fluid dispensing devicesknown in the art, since it provides for enhanced accuracy, precision,and efficiency in fluid dispensing through the use of a means foracoustically determining one or more properties of the fluid withinreservoirs and/or the reservoir materials themselves.

As discussed above, acoustic ejection provides a number of advantagesover other fluid dispensing technologies. An additional advantage of theinvention is that it provides a device that can carry out both acousticejection and assessment. In such a case, the acoustic radiationgenerator may serve as a component of both an acoustic ejector and anacoustic assessing means. For example, compatible acoustic ejectiontechnology described in U.S. Ser. No. 09/964,212 involves an ejectorcomprising an acoustic radiation generator for generating acousticradiation and a focusing means for focusing the acoustic radiationgenerated at a focal point within and sufficiently near the fluidsurface in each of a plurality of reservoirs to result in the ejectionof droplets therefrom.

Optionally, a focusing means is typically provided for focusing theacoustic radiation generated by the acoustic generator. In the presentinvention, any of a variety of focusing means may be employed inconjunction with the acoustic generator in order to eject droplets froma reservoir through the use of focused acoustic radiation. For example,one or more curved surfaces may be used to direct acoustic radiation toa focal point near a fluid surface. One such technique is described inU.S. Pat. No. 4,308,547 to Lovelady et al. Focusing means with a curvedsurface have been incorporated into the construction of commerciallyavailable acoustic transducers, such as those manufactured byPanametrics Inc. (Waltham, Mass.). In addition, Fresnel lenses are knownin the art for directing acoustic energy at a predetermined focaldistance from an object plane. See, e.g., U.S. Pat. No. 5,041,849 toQuate et al. Fresnel lenses may have a radial phase profile thatdiffracts a substantial portion of acoustic energy into a predetermineddiffraction order at diffraction angles that vary radially with respectto the lens. The diffraction angles should be selected to focus theacoustic energy within the diffraction order on a desired object plane.Optimally, the device is adapted to eject fluid from a reservoiraccording to the results of acoustic analysis performed by the analyzer.

The device may also provide certain performance-enhancingfunctionalities. For example, the device may include a means forcontrolling the temperature of one or more of the reservoirs. Suchtemperature controlling means may be employed in the inventive device toimprove the accuracy of measurement, and may be employed regardless ofwhether the device includes a fluid dispensing functionality. In thecase of aqueous fluids, the temperature controlling means should havethe capacity to maintain the reservoirs at a temperature above about 0°C. In addition, the temperature controlling means may be adapted tolower the temperature in the reservoirs. Such temperature lowering maybe required because repeated application of acoustic energy to areservoir of fluid may result in heating of the fluid. Such heating canresult in unwanted changes in fluid properties, such as viscosity,surface tension, and density. Design and construction of suchtemperature controlling means are known to one of ordinary skill in theart and may comprise, e.g., components such as a heating element, acooling element, or a combination thereof. For many biomolecularapplications, reservoirs of fluids are stored frozen and thawed for use.During use, it is generally desired that the fluid containing thebiomolecule be kept at a constant temperature, with deviations of nomore than about 1 or 2° C. therefrom. In addition, for a biomolecularfluid that is particularly heat sensitive, it is preferred that thefluid be kept at a temperature that does not exceed about 10° C. abovethe melting point of the fluid, preferably at a temperature that doesnot exceed about 5° C. above the melting point of the fluid. Thus, forexample, when the biomolecule-containing fluid is aqueous, it may beoptimal to keep the fluid at about 4° C. during ejection. It isdesirable to measure the temperature in the fluid, or to infer thistemperature from the measured temperature of the reservoir containingit, to provide input to a thermal controller and to use in thedetermination of acoustic parameters that may be temperature dependent.

Moreover, the device may be adapted to assess and/or dispense fluids ofvirtually any type and amount desired. The fluid may be aqueous and/ornon-aqueous. Examples of fluids include, but are not limited to, aqueousfluids including water per se and water-solvated ionic and non-ionicsolutions, organic solvents, lipid liquids, mixtures of immisciblefluids, and suspensions or slurries of solids in liquids. Because theinvention is readily adapted for use with high temperatures, fluids suchas liquid metals, ceramic materials, and glasses may be used; see, e.g.,U.S. Pat. No. 6,548,308 to Ellson and Mutz for “Method and Apparatus forGenerating Droplets of Immiscible Fluids.” Furthermore, because of theprecision that is possible using the inventive technology, the devicemay be used to eject droplets from a reservoir adapted to contain nomore than about 100 nanoliters of fluid, preferably no more than 10nanoliters of fluid. In certain cases, the ejector may be adapted toeject a droplet from a reservoir adapted to contain about 1 to about 100nanoliters of fluid. This is particularly useful when the fluid to beejected contains rare or expensive substances, wherein it may bedesirable to eject droplets having a volume of about 1 picoliter (pL) orless, e.g., having a volume in the range of about 0.025 pL to about 1pL.

Thus, another embodiment of the invention relates to a method fordispensing fluid from one or more reservoirs. Once an acoustic radiationgenerator is positioned in acoustic coupling relation to a reservoirselected from a plurality of reservoirs, acoustic radiation generated bythe acoustic radiation generator may be transmitted through at least aportion of the selected reservoir. The acoustic radiation is thenanalyzed in order to determine the properties of the contents of thereservoir, and fluid is dispensed from the selected reservoir accordingto that determination. Typically, the fluid is dispensed throughacoustic ejection, though the inventive method may employ contact-basedfluid dispensing, either as an alternative to or as a supplement tonon-contact fluid dispensing. Optionally, the above process may berepeated for additional reservoirs.

It should be noted that there are a number of different ways to combineacoustic evaluation of fluid properties with fluid dispensing, dependingon the intended purpose of the combination. As discussed above, fluidmay be dispensed from a reservoir after the properties of the fluid inthe reservoir have been acoustically determined. This allows an operatorto fine-tune the dispensing according to the properties of the contentsof the reservoir. In addition, fluid may be dispensed from a reservoirbefore the properties of the contents of the reservoir are acousticallyassessed. In such a case, acoustic evaluation may serve to confirm thequality of fluid dispensation as well as to ensure that the dispensingprocess does not unexpectedly alter the contents of the reservoir. Insome instances, acoustic assessment and fluid dispensation may occursimultaneously.

FIG. 1 illustrates a preferred embodiment of the inventive device insimplified cross-sectional view. In this embodiment, the inventivedevice allows for acoustic assessment of the contents of a plurality ofreservoirs as well as acoustic ejection of fluid droplets from thereservoirs. The inventive device is shown in operation to form abiomolecular array bound to a substrate. As with all figures referencedherein, in which like parts are referenced by like numerals, FIG. 1 isnot to scale, and certain dimensions may be exaggerated for clarity ofpresentation. The device 11 includes a plurality of reservoirs, i.e., atleast two reservoirs, with a first reservoir indicated at 13 and asecond reservoir indicated at 15. Each reservoir may be contained in, ordisposed on, a substrate, such as well plate 111 (shown in FIG. 4A).Each is adapted to contain a fluid having a fluid surface. As shown, thefirst reservoir 13 contains a first fluid 14, and the second reservoir15 contains a second fluid 16. Fluids 14 and 16 each have a fluidsurface, respectively indicated at 17 and 19. Fluids 14 and 16 may bethe same or different. As shown, the reservoirs are of substantiallyidentical construction so as to be substantially acousticallyindistinguishable, but identical construction is not a requirement. Thereservoirs are shown as separate removable components but may, asdiscussed above, be fixed within a plate or other substrate. Forexample, the plurality of reservoirs may comprise individual wells in awell plate, optimally although not necessarily arranged in an array.Each of the reservoirs 13 and 15 is preferably axially symmetric asshown, having vertical walls 21 and 23 extending upward from circularreservoir bases 25 and 27 and terminating at openings 29 and 31,respectively, although other reservoir shapes may be used. The materialand thickness of each reservoir base should be such that acousticradiation may be transmitted therethrough and into the fluid containedwithin the reservoirs.

The device also includes an acoustic ejector 33 comprised of an acousticradiation generator 35 for generating acoustic radiation and a focusingmeans 37 for focusing the acoustic radiation at a focal point within thefluid from which a droplet is to be ejected, near the fluid surface. Theacoustic radiation generator 35 contains a transducer 36, e.g., apiezoelectric element, commonly shared by an analyzer. As shown, acombination unit 38 is provided that serves both as a controller and acomponent of an analyzer. Operating as a controller, the combinationunit 38 provides the piezoelectric element 36 with electrical energythat is converted into mechanical and acoustic energy. Operating as acomponent of an analyzer, the combination unit receives and analyzeselectrical signals from the transducer. The electrical signals areproduced as a result of the absorption and conversion of mechanical andacoustic energy by the transducer.

As shown in FIG. 1, the focusing means 37 may comprise a single solidpiece having a concave surface 39 for focusing acoustic radiation, butthe focusing means may be constructed in other ways as discussed below.The acoustic ejector 33 is thus adapted to generate and focus acousticradiation so as to eject a droplet of fluid from each of the fluidsurfaces 17 and 19 when acoustically coupled to reservoirs 13 and 15,and thus to fluids 14 and 16, respectively. The acoustic radiationgenerator 35 and the focusing means 37 may function as a single unitcontrolled by a single controller, or they may be independentlycontrolled, depending on the desired performance of the device.Typically, single ejector designs are preferred over multiple ejectordesigns because accuracy of droplet placement and consistency in dropletsize and velocity are more easily achieved with a single ejector.

There are a number of ways to acoustically couple the ejector 33 to eachindividual reservoir and thus to the fluid therein. One such approach isthrough direct contact as is described, for example, in U.S. Pat. No.4,308,547 to Lovelady et al., wherein a focusing means constructed froma hemispherical crystal having segmented electrodes is submerged in aliquid to be ejected. The aforementioned patent further discloses thatthe focusing means may be positioned at or below the surface of theliquid. However, this approach for acoustically coupling the focusingmeans to a fluid is undesirable when the ejector is used to ejectdifferent fluids in a plurality of containers or reservoirs, as repeatedcleaning of the focusing means would be required in order to avoidcross-contamination. The cleaning process would necessarily lengthen thetransition time between each droplet ejection event. In addition, insuch a method, fluid would adhere to the ejector as it is removed fromeach container, wasting material that may be costly or rare.

Thus, a preferred approach would be to acoustically couple the ejector33 to the reservoirs 13, 15 and reservoir fluids 14, 16 withoutcontacting any portion of the ejector 33, e.g., the focusing means 37,with any of the fluids to be ejected. To this end, the present inventionprovides an ejector positioning means 43 for positioning the ejector 33in controlled and repeatable acoustic coupling with each of the fluids14, 16 in the reservoirs 13, 15 to eject droplets therefrom withoutsubmerging the ejector 33 therein. This typically involves direct orindirect contact between the ejector 33 and the external surface of eachreservoir 13, 15. When direct contact is used in order to acousticallycouple the ejector 33 to each reservoir 13, 15, it is preferred that thedirect contact be wholly conformal to ensure efficient acoustic energytransfer. That is, the ejector 33 and the each of the reservoirs 13, 15should have corresponding surfaces adapted for mating contact. Thus, ifacoustic coupling is achieved between the ejector 33 and one of thereservoirs 13, 15 through the focusing means 37, it is desirable for thereservoir 13 or 15 to have an outside surface that corresponds to thesurface profile of the focusing means 37. Without conformal contact,efficiency and accuracy of acoustic energy transfer may be compromised.In addition, since many focusing means have a curved surface, the directcontact approach may necessitate the use of reservoirs having aspecially formed inverse surface.

Optimally, acoustic coupling is achieved between the ejector 33 and eachof the reservoirs 13, 15 through indirect contact, as illustrated inFIG. 1A. In this figure, an acoustic coupling medium 41 is placedbetween the ejector 33 and the base 25 of reservoir 13, with the ejector33 and reservoir 13 located at a predetermined distance from each other.The acoustic coupling medium 41 may be an acoustic coupling fluid,preferably an acoustically homogeneous material in conformal contactwith both the acoustic focusing means 37 and each reservoir. Inaddition, it is important to ensure that the coupling medium 41 issubstantially free of material having different acoustic properties thanthe coupling fluid medium itself. Furthermore, it is preferred that theacoustic coupling medium is comprised of a material having acousticproperties that facilitate the transmission of acoustic radiationwithout significant attenuation in acoustic intensity. Also, theacoustic impedance of the coupling medium 41 should facilitate thetransfer of energy from the coupling medium 41 into the reservoir 13,and the coupling medium 41 should remain a liquid over the range ofinstrument operating temperatures. As shown, the first reservoir 13 isacoustically coupled to the acoustic focusing means 37, such that anacoustic wave is generated by the acoustic radiation generator 35 anddirected by the focusing means 37 into the acoustic coupling medium 41,which then transmits the acoustic radiation into the reservoir 13.

In operation, reservoirs 13 and 15 are each filled with first and secondfluids 14 and 16, respectively, as shown in FIG. 1. The acoustic ejector33 is positionable by means of ejector positioning means 43, shown belowreservoir 13, in order to achieve acoustic coupling between the ejector33 and the reservoir 13 through acoustic coupling medium 41. Once theejector 33, the reservoir 13, and the substrate 45 are in properalignment, the acoustic radiation generator 35 is activated to produceacoustic radiation that is directed toward a free fluid surface 17 ofthe first reservoir 13. The acoustic radiation then travels in agenerally upward direction toward the free fluid surface 17. Theacoustic radiation is reflected under different circumstances.Typically, reflection occurs when there is a change in the acousticproperty of the medium through which the acoustic radiation istransmitted. We have observed that a portion of the acoustic radiationtraveling upward is reflected by the reservoir bases 25 and 27 as wellas by the free surfaces 17 and 19 of the fluids contained in thereservoirs 13 and 15.

Thus, the present invention represents a significant improvement overknown technologies relating to the acoustic assessment of the contentsof reservoirs. As discussed above, acoustic assessment of the contentsof liquid reservoirs typically involves placing a sensor in directcontact with the liquid. This means that the sensor must be cleanedbetween each use to avoid cross-contamination of the contents of thereservoirs. In contrast, the present invention allows for assessment ofthe contents of a plurality of containers without direct contact withthe contents of the containers.

While other non-contact acoustic systems are known in the art, suchsystems provide only an indirect and approximate assessment of thecontents of a reservoir. For example, the acoustic system described inU.S. Pat. No. 5,880,364 to Dam employs a technique in which the acousticradiation is transmitted from a sensor through an air-containing portionof the container and then reflected from the air-liquid interface of thecontainer back to the sensor. The roundtrip transit time is used todetermine the volume of the air-containing portion of the container. Thevolume of liquid in the container is determined by subtracting thevolume of the container not occupied by the liquid from the volume ofthe entire container. One drawback of this technique is that it cannotprovide an accurate assessment of the liquid volume in a container whenthe volume of the container is not precisely known. This is particularlyproblematic when small reservoirs, such as those typically used incombinatorial techniques, are employed. The dimensional variability forsuch containers is relatively large when considered in view of the smallvolume of the reservoirs. Furthermore, the technique cannot be employedwhen the volume of the container is completely unknown or alterable.Finally, since acoustic radiation never penetrates the liquid, thereflected radiation can at best only provide information relating to thesurface of the liquid, not information relating to the bulk of theliquid.

In contrast, because the invention involves the transmission of acousticradiation through the portion of each reservoir adapted to contain afluid, the transmitted acoustic radiation may provide informationrelating to the volume as well as the properties of the fluids in thereservoir. For example, the invention may provide a plurality ofreservoirs, wherein a portion of each reservoir is adapted to containfluid. A fluid contained in a reservoir must ordinarily contact a solidsurface of the reservoir. When the invention is employed in a reflectivemode, some of the generated acoustic radiation may be reflected by theinterface between the fluid and the solid surface, while the remainderis transmitted through a fluid contained in the reservoir. Thetransmitted radiation is then reflected by another surface, e.g., a freesurface, of the fluid contained in the reservoir. By determining thedifference in roundtrip transit times between the two portions, thevolume of the fluid in the reservoir may be accurately determined. Inaddition, transmission of acoustic radiation through the fluid allowscharacteristics of the acoustic radiation to be altered by the fluid.Thus, information relating to a property of the fluid may be deduced byanalyzing a characteristic of the transmitted acoustic radiation.

In addition, air, like other gases, exhibits low acoustic impedance, andacoustic radiation tends to attenuate more in gaseous materials than inliquid or solid materials. For example, the attenuation at 1 MHz for airis approximately 10 dB/cm while that of water is 0.002 dB/cm. Since theacoustic system described in U.S. Pat. No. 5,880,364 to Dam requiresacoustic radiation to travel through air, this system requires much moreenergy to operate. Thus, the present invention represents a more energyefficient technology, which may be employed to provide a more accurateand detailed assessment of the contents of a plurality of fluidreservoirs. Some of this additional accuracy can be achieved by usingacoustic waves with higher frequencies (and hence shorter wavelengths),as these acoustic waves can be transmitted effectively through liquidsyet would be very rapidly attenuated in air.

It will be appreciated by those of ordinary skill in the art thatconventional or modified sonar techniques may be employed in thepractice of this invention. Thus, acoustic (sonar) radiation isreflected back to the piezoelectric element 36, where the acousticenergy is converted into electrical energy for analysis. The analysismay be used, for example, to reveal whether the reservoir contains anyfluid at all. If fluid is present in the reservoir, the location and theorientation of the free fluid surface within the reservoir may bedetermined, as well as the overall volume of the fluid. Characteristicsof the reflected acoustic radiation may be analyzed in order to assessthe spatial relationship between the acoustic radiation generator andthe fluid surface, the spatial relationship between a solid surface ofthe reservoir and the fluid surface, as well as to determine a propertyof the fluid in each reservoir, e.g., viscosity, surface tension,temperature, acoustic impedance, acoustic attenuation, solid content,and impurity content. Once the analysis has been performed, a decisionmay be made as to whether and/or how to dispense fluid from thereservoir.

Depending on the type of assessment to be carried out, varioustechniques known in the art may be adapted for use in the presentinvention. Generally, interfacial energy measurements are routinelycarried out using contact-angle measurement. The present invention maybe adapted to perform such contact-angle measurements. In addition, anumber of other acoustic assessment techniques are known in the art. Forexample, U.S. Pat. No. 4,391,129 to Trinh describes a system formonitoring the physical characteristics of fluids. The physicalcharacteristics may be determined from acoustic assessment of theinterfacial tension of fluids to a high degree of accuracy. U.S. Pat.No. 4,558,589 to Hemmes describes an ultrasonic blood-coagulationmonitor. U.S. Pat. No. 5,056,357 to Dymling et al. describes acousticmethods for measuring properties in fluids through Doppler shifts. Otheracoustic assessment techniques that may be adapted for use in thepresent invention are described, for example, in U.S. Pat. Nos.4,901,245; 5,255,564; 5,410,518; 5,471,872; 5,533,402; 5,594,165;5,623,095; 5,739,432; 5,767,407; 5,793,705; 5,804,698; 6,119,510;6,227,040; and 6,298,726.

In order to form a biomolecular array on a substrate using the inventivedevice, substrate 45 is positioned above and in proximity to the firstreservoir 13 such that one surface of the substrate, shown in FIG. 1 asunderside surface 51, faces the reservoir and is substantially parallelto the surface 17 of the fluid 14 therein. Once the ejector, thereservoir, and the substrate are in proper alignment, the acousticradiation generator 35 is activated to produce acoustic radiation thatis directed by the focusing means 37 to a focal point 47 near the fluidsurface 17 of the first reservoir. That is, an ejection acoustic wavehaving a focal point near the fluid surface is generated in order toeject at least one droplet of the fluid, wherein the optimum intensityand directionality of the ejection acoustic wave is determined using theaforementioned analysis, optionally in combination with additional data.The “optimum” intensity and directionality are generally selected toproduce droplets of consistent size and velocity. For example, thedesired intensity and directionality of the ejection acoustic wave maybe determined by using the data from the above-described assessmentrelating to reservoir volume or fluid property data, as well asgeometric data associated with the reservoir. In addition, the data mayshow the need to reposition the ejector so as to reposition the acousticradiation generator with respect to the fluid surface, in order toensure that the focal point of the ejection acoustic wave is near thefluid surface, where desired. For example, if analysis reveals that theacoustic radiation generator is positioned such that the ejectionacoustic wave cannot be focused near the fluid surface, the acousticradiation generator is repositioned using vertical, horizontal, and/orrotational movement to allow appropriate focusing of the ejectionacoustic wave.

As a result, droplet 49 is ejected from the fluid surface 17 onto adesignated site on the underside surface 51 of the substrate. Theejected droplet may be retained on the substrate surface by solidifyingthereon after contact; in such an embodiment, it may be necessary tomaintain the substrate at a low temperature, i.e., a temperature thatresults in droplet solidification after contact. Alternatively, or inaddition, a molecular moiety within the droplet attaches to thesubstrate surface after contact, through adsorption, physicalimmobilization, or covalent binding.

Then, as shown in FIG. 1B, a substrate positioning means 50 repositionsthe substrate 45 over reservoir 15 in order to receive a droplettherefrom at a second designated site. FIG. 1B also shows that theejector 33 has been repositioned by the ejector positioning means 43below reservoir 15 and is in an acoustically coupled relationshipthereto by virtue of acoustic coupling medium 41. Once properly aligned,the acoustic radiation generator 35 of ejector 33 is activated toproduce low energy acoustic radiation to assess the contents of thereservoir 15 and to determine whether and/or how to eject fluid from thereservoir. Historical droplet ejection data associated with the ejectionsequence may be employed as well. Again, there may be a need toreposition the ejector so as to reposition the acoustic radiationgenerator with respect to the fluid surface, in order to ensure that thefocal point of the ejection acoustic wave is near the fluid surface,where desired. Should the results of the assessment indicate that fluidmay be dispensed from the reservoir, focusing means 37 is employed todirect higher energy acoustic radiation to a focal point 48 within fluid16 near the fluid surface 19, thereby ejecting droplet 53 onto thesubstrate 45.

It will be appreciated that various components of the device may requireindividual control or synchronization to form an array on a substrate.For example, the ejector positioning means may be adapted to ejectdroplets from each reservoir in a predetermined sequence associated withan array to be prepared on a substrate surface. Similarly, the substratepositioning means for positioning the substrate surface with respect tothe ejector may be adapted to position the substrate surface to receivedroplets in a pattern or array thereon. Either or both positioningmeans, i.e., the ejector positioning means and the substrate positioningmeans, may be constructed from, for example, motors, levers, pulleys,gears, a combination thereof, or other electromechanical or mechanicalmeans known to one of ordinary skill in the art. It is preferable toensure that there is a correspondence between the movement of thesubstrate, the movement of the ejector, and the activation of theejector to ensure proper array formation.

Accordingly, the invention relates to the assessment of the contents ofa plurality of reservoirs as well as to dispensing a plurality of fluidsfrom reservoirs, e.g., in order to form a pattern or an array, on thesubstrate surface 51. However, there are a number of different ways inwhich content assessment and fluid dispensing may relate to each other.That is, a number of different sequences may be employed for assessingthe contents of the reservoirs and for dispensing fluids therefrom. Insome instances, the contents of a plurality of reservoirs may beassessed before fluid is dispensed from any of the reservoirs. In otherinstances, the contents of each reservoir may be assessed immediatelybefore fluid is dispensed therefrom. The sequence used typically dependson the particular fluid-dispensing technique employed as well as theintended purpose of the sequence.

FIG. 2 illustrates an example of the inventive device that provides forassessment of the contents of a plurality of reservoirs in transmissivemode rather than in reflective mode. Considerations for the design andconstruction of this device are similar to those discussed above. Thus,the device 11 includes a first reservoir 13 and a second reservoir 15,each adapted to contain a fluid indicated at 14 and 16, respectively,and each of substantially identical construction. The first reservoir 13is depicted in an open state, while the second reservoir is depicted ina sealed state. An acoustic radiation generator 35 is positioned belowthe reservoirs, and analyzer 38 is positioned in opposing relationshipwith the acoustic radiation generator 35 above the reservoirs.

In operation, the contents of each of the reservoirs are acousticallyevaluated before pipette 60 is employed to dispense fluid therefrom. Asshown, the contents 14 of the first reservoir 13 have already beenacoustically assessed. As the assessment has revealed that the firstreservoir 13 contains at least a minimum acceptable level of fluid 14,the first reservoir 13 is open and ready for fluid to be dispensedtherefrom via pipette 60. The contents 16 of the second reservoir 15 areundergoing acoustic assessment, as depicted by FIG. 2, as the secondreservoir 15 is interposed between the acoustic radiation generator 35and the analyzer 38. The acoustic radiation generator 35 and theanalyzer 38 are acoustically coupled to the second reservoir viacoupling media 41 and 42, respectively. Once the acoustic radiationgenerator 35, the second reservoir 15, and the analyzer 38 are in properalignment, the acoustic radiation generator 35 is activated to produceacoustic radiation that is transmitted through the reservoir 15 and itscontents 16 toward the analyzer 38. The received acoustic radiation isanalyzed by an analyzer 38, as described above.

The present invention can be used to determine the bulk temperature ofmaterials. When the material between two interfaces is known and thedistance the acoustic pulse travels in this material is also known, thespeed of sound in the material can be determined by dividing thedistance traveled by the travel time. The speed of sound in manymaterials changes monotonically with temperature, and hence knowledge ofthe speed of sound in the material is an indicator of the averagetemperature along the path of the acoustic wave within the material.

Thus the transit time for sound through a well plate bottom can be usedas a thermometer for the microplate at a given well location. The speedof sound changes sufficiently as a function of temperature for mostmaterials so that a temperature change of 1° C. can be detected with a500 MHz signal, for a typical microplate thickness of about 500 μm. Theresulting sensitivity is well in excess of that for the case discussedsupra, in which the sampling time was required to be at least 100 timesthe transit time. For example, a 500 MHz signal has a sampling time of 2ns. A typical transit time for 500 μm of polypropylene is about 710 ns.Therefore, the sampling time is about 355 times (710 ns/2 ns) thetransit time. One can thus detect changes on the order of 1/355. For amaterial like polypropylene, with about a 0.3% change in the speed ofsound per degree Celsius, one should obtain measurements accurate towithin about 1° C.

FIG. 3 shows a graph for properties associated with a DMSO/water mixturein a sealed microplate that is warmed to room temperature. Elapsed time(hh:mm:ss) is shown along the x-axis. On the left-hand y-axis, themeasured apparent DMSO content in a DMSO/water mixture is shown. On theright-hand y-axis, the well bottom travel time is shown in seconds. Inthe upper curve of FIG. 3, for example, the transit time of an acousticwave through a polypropylene microplate (an average time for all 384wells) is shown to increase for about an hour, settling at a value near0.71 μsec.

The increase in travel time through the well plate bottom as it warms isdue to the reduction in the speed of sound as a function of temperaturefor polypropylene. This response is typical for polypropylene materials.As discussed supra, Profax Polypropylene shows a 0.342% reduction in thespeed of sound per ° C. (at temperatures close to room temperature).Thus, if a Profax Polypropylene plate experiences a 10% reduction in thespeed of sound as it warmed to room temperature, an approximate 30° C.warming of the microplate bottom is indicated. In general, if thetransit time can be measured through a plate that is made of a materialfor which the speed of sound is a known function of temperature, thenthe plate can act as a thermometer. The transit time gives a “bulktemperature” based on the average velocity of sound through the plate(e.g., well bottom). If the “bulk temperature” is representative of thefluid/plate interface temperature, then accurate values of impedance forthe materials can be used to determine the reflected echo amplitude.From such data, a temperature-corrected/compensated impedance model, andthus reflection amplitude curve, can be constructed or empiricallyderived, as discussed supra.

FIG. 4 diagrammatically shows the principles associated with the use ofthe speed of sound as a parameter to determine the composition of afluid in a well.

In FIG. 4A a well 113 in a well plate 114 has a base 115 and sidewalls117. A fluid 119 is disposed in the well 113. An acoustic wave 121,generated by an acoustic wave generator 35, engages an interface 123between the base 115 of the well 113 and the fluid 119 in the well 113.Because there is a substantial difference between the acoustic impedanceZf (T) of the fluid 119 and the acoustic impedance Zp (T) of the base115, the acoustic wave 121 is reflected back to the analyzer provided incombination unit 38 for processing.

The travel time of the acoustic wave 121 inside the base 115 changeswith temperature as the speed of sound inside the plate (cp) changeswith temperature. Therefore, the bulk plate temperature can becalculated from the travel time, determined by a comparison of the timebetween the engagements of the acoustic wave with a lower surface 115 aof the base 115 and the base/fluid interface 123. The arrival time atthe transducer of the acoustic wave reflected from the two surfacesrepresents the time it takes for a round trip in the base.

FIG. 4B shows that the acoustic impedance of the well fluid 119 and thewell 113 change at different rates with changes in temperature. That is,as temperature rises, the impedance of fluid 119 in the well 113 mayrise faster than the impedance of the well material because of theinherent difference in acoustic impedance between the fluid and the wellmaterial. This will change the amplitude of the reflected wave 121 atthe base/fluid interface 123. This change could suggest a change in theDMSO composition of fluid that is, in fact, erroneous. Any modeldeveloped to use temperature to determine fluid composition would haveto recognize and compensate for the phenomenon shown in FIG. 4B.

Because a single reading at the well base 115 may give an erroneousreading of temperature for the well 113 and the plate 111, it would bedesirable to take multiple temperature readings in the plate 111 and thewell 113. An example of this approach is shown in FIG. 4C. Bulk platetemperature can be determined at different locations in the plate 111with respect to fluid 119. Multiple reflected transit times can be takenin the plate 111, the base 115 of the well 113, as well as the sidewalls117. FIG. 4D, a top view of the well 113, shows test points 113 athrough 1131, used to obtain transit times employed for temperaturemeasurements.

FIG. 5 provides a computer model for analyzing acoustic data tocalculate DMSO content in a DMSO/water mixture with the use oftemperature determination and correction. In step 201 the reflectedsignal is processed for time-of-flight and amplitude. From these datathe plate bottom transit time is extracted (Step 203). In step 205 theplate bottom transit reference time is retrieved. In step 207, data areretrieved for the speed of sound as a function of temperature for theplate material. The data retrieved in steps 203, 205, and 207 are thendelivered to a computer model 209, to convert reflection amplitude toDMSO content based on the acoustic impedance, Z(T), for the materialsanalyzed. The output of the model 209 is step 211, DMSO content.

Hence, the acoustic impedance change with temperature for both themicroplate and well fluid (or solid if the temperature is below thefreezing point of the liquid) would be corrected over a broadtemperature range (−80° C. up to a “warm” room temperature).

Since pulse timing can be determined with great precision, by samplingor digitizing the echo return signal of the acoustic pulse, this methodof “acoustic thermometry” can be implemented with the same electronicsas is applied to other acoustic measurements for materialcharacteristics.

Thicknesses for materials in reservoir containers vary with the materialand reservoir used. Microplates used for optical assays typically havefilm bottoms of between 50 and 200 μm and may be composed of polystyreneor cyclic olefin copolymer. Microplates and tubes used for compoundlibrary storage have thicker bottoms, typically between 0.5 and 1.2 mm,and may be composed of polypropylene, cyclic olefin copolymer, or glass.

In general, the path of travel for the acoustic pulse directed withinthe walls of reservoirs will be larger that the travel distance withinthe bottom of a reservoir. Walls for standard microplates range from 2mm to 12 mm in height, and so-called “deep wells” may possess walls withheights in excess of 25 mm. Storage tubes and mini tubes may range insize from 8 mm to over 25 mm. Since longer transit distances producegreater absolute differences in transit times as a function oftemperature, it is easier to make accurate bulk temperature measurementsin thicker portions of the reservoir.

The speed of sound as a function of temperature in materials can bedetermined by a variety of techniques known to those of skill in theart. For example, the speed of sound can be found empirically byimmersing a sample of the material with a known thickness in a bath ofcontrolled temperature, and then measuring the acoustic wave travel timethrough the material. Polymeric materials found in microplates showsignificant changes in sound speed as a function of temperature. Forexample, the speed of sound in some polypropylenes changes more than0.3% per degree C. at temperatures near room temperature (22° C.). Thespeed of sound in polystyrenes changes about 0.1% per degree C. Thesevalues are approximate, since the wide range of molecular weightdistributions and processing methods for these polymers influence thespeed of sound. The speed of sound can vary with temperature by over 50%from these values, yet for a particular microplate, made with aparticular polymer formulation and molding process, the speed of soundas a function of temperature is much more consistent at a givenmicroplate well, and can serve as the basis for acoustic thermometry.

One temperature measurement of interest is that of the fluid in thereservoir, and the reservoir fluid may not be of either a knowncomposition or a known depth. One object of the present invention is todetermine the temperature of the fluid by inference from the temperatureof the reservoir material around it. As shown in FIGS. 4A-4C, the speedof sound can be measured in both the reservoir bottom and the walls ofthe reservoir. These walls can be either the walls of a microtube or theseparator walls of wells in a multi-well container such as a microplate.An average temperature along the path of the acoustic beam is collectedby sending the beam into a given location. As discussed supra, to builda better spatial profile of the average temperature around a givenreservoir fluid, acoustic pulses can be directed to multiple differentlocations, including some points located at the bottom of the fluid welland others within the reservoir wall.

It should be evident, then, that the invention provides a number ofpreviously unrealized advantages for assessing the contents of aplurality of reservoirs. First, acoustic assessment is a generallynoninvasive technique that may be carried out regardless of whether thereservoirs are sealed or open. That is, acoustic assessment does notrequire extracting a sample for analysis or require other mechanicalcontact that may result in sample cross-contamination. In addition,unlike optical detection techniques, optically translucent ortransparent reservoirs are not required. This, of course, provides awider range of choices for materials that may be employed for reservoirconstruction. In addition, the use of opaque material would beparticularly advantageous in instances wherein the reservoirs areconstructed to contain photosensitive fluids.

Thus, variations of the present invention will be apparent to those ofordinary skill in the art. For example, while FIG. 1 depicts theinventive device in operation to form a biomolecular array bound to asubstrate, the device may be operated in a similar manner to format aplurality of fluids, e.g., to transfer fluids from odd-sized bulkcontainers to wells of a standardized well plate. Similarly, while FIG.2 illustrates that the acoustic radiation generator and the detector arein vertical opposing relationship, other spatial and/or geometricarrangements may be employed so long as acoustic radiation generated istransmitted through at least a portion of the reservoir to the detector.

As another example, the invention may be employed to detect whether thecontents of a sealed reservoir are at least partially frozen, withoutopening the reservoir. This would be useful when it is known that areservoir contains a substance that is capable of existing as a fluidover a certain temperature range, but it is unclear as to thetemperature history of the reservoir, e.g., whether freeze/thaw cyclesexisted. For example, water is capable of existing as a fluid at atemperature of about 0° C. to about 100° C. If it is unclear whether theexterior temperature of a reservoir is indicative of the reservoir'sinterior temperature, but the reservoir is known to contain liquidwater, the inventive device is well suited to determine whether any orall of the contents of the reservoir is a fluid.

In addition, the invention may be constructed to be highly compatiblewith existing infrastructure of materials discovery and with existingautomation systems for materials handling. For example, the inventionmay be adapted for use as an alternative or a supplement to contentassessment means that are based on optical detection. In some instances,sonic markers may be provided in the reservoirs to identify the contentsof the reservoir. Thus, the invention may be employed as a means forinventory identification and control in a number of contexts, including,but not limited, to biological, biochemical, and chemical discovery andanalysis.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, journal articles, and other referencescited herein are incorporated by reference in their entireties.

1. A method for acoustically determining a temperature-dependentproperty of a fluid in a reservoir, the reservoir being integral with orlocated on a substrate, the method comprising: (a) acoustically couplingan acoustic radiation generator and the reservoir, with the acousticradiation generator located external to the reservoir; (b) activatingthe acoustic radiation generator to generate acoustic radiation anddirect the acoustic radiation generated into and through the substratetoward the reservoir, such that the acoustic radiation is transmittedinto the fluid in the reservoir and thereafter reflected from aninterior surface of the reservoir; (c) measuring a temperature-dependentcharacteristic of the reflected acoustic radiation, said characteristicselected so as to correlate with the temperature of the fluid; and (d)employing the characteristic to determine the temperature-dependentproperty of the fluid in a temperature-compensated manner.
 2. The methodof claim 1, wherein the interior surface of the reservoir is aninterface between the fluid and an interior surface of the reservoir. 3.The method of claim 2, wherein the temperature (T_(F1)) of the fluid islower than 20° C. or greater than 25° C.
 4. The method of claim 3,wherein the temperature-dependent characteristic of the reflectedacoustic radiation is the acoustic velocity V_(F1) in the fluid attemperature T_(F1).
 5. The method of claim 4, wherein (d) comprisescomparing V_(F1) with the acoustic velocity V_(F-RT) of the fluid at atemperature T_(RT) in the range of 20° C. to 25° C.
 6. The method ofclaim 5, wherein (d) further comprises deriving from the differencebetween V_(F1) and V_(F-RT) the difference between the acousticimpedance Z_(F1) of the fluid at temperature T_(F1) and the acousticimpedance Z_(F-RT) of the fluid at temperature T_(RT).
 7. The method ofclaim 6, wherein the temperature-dependent property is the compositionof the fluid, and (d) further comprises determining the composition ofthe fluid in the reservoir from Z_(F1) and Z_(F-RT).
 8. The method ofclaim 7, wherein the fluid in the reservoir is at least partiallynonsolid.
 9. The method of claim 7, wherein the fluid in the reservoircomprises at least one liquid.
 10. The method of claim 9, wherein thefluid in the reservoir comprises a combination of miscible, partiallymiscible, or immiscible liquids.
 11. The method of claim 10, wherein thefluid in the reservoir comprises a mixture of an organic solvent and anaqueous solvent.
 12. The method of claim 11, wherein the fluid in thereservoir comprises a mixture of dimethylsulfoxide (DMSO) and water. 13.The method of claim 12, wherein the mixture contains more than 40 vol. %DMSO.
 14. The method of claim 12, wherein the mixture contains more than40 vol. % water.
 15. The method of claim 1, wherein the reservoir isintegral with a substrate.
 16. The method of claim 15, wherein thereservoir is an individual container.
 17. The method of claim 16,wherein the container is enclosed.
 18. The method of claim 16, whereinthe reservoir is a tube.
 19. The method of claim 1, wherein thereservoir is one of a plurality of reservoirs, each reservoir beingintegral with or located on a substrate.
 20. The method of claim 19,wherein the reservoirs are integral with a substrate.
 21. The method ofclaim 20, wherein the substrate is a well plate and each reservoir is awell therein.
 22. The method of claim 19, wherein steps (a) through (d)are repeated successively for additional reservoirs.
 23. The method ofclaim 22, wherein steps (a) through (d) are repeated successively foreach additional reservoir.
 24. The method of claim 23, wherein steps (a)through (d) are repeated at a rate of at least 96 reservoirs per minute.25. The method of claim 24, wherein steps (a) through (d) are repeatedat a rate of at least about 384 reservoirs per minute.
 26. The method ofclaim 25, wherein steps (a) through (d) are repeated at a rate of atleast about 1536 reservoirs per minute.
 27. The method of claim 26,wherein steps (a) through (d) are repeated at a rate of at least about3456 reservoirs per minute.
 28. The method of claim 27, wherein steps(a) through (d) are repeated at a rate of at least 10,000 reservoirs perminute.
 29. The method of claim 1, wherein the acoustic radiationgenerated is directed into the reservoir through a region of thereservoir's exterior surface having an identifier thereon.
 30. Themethod of claim 29, wherein the identifier is a barcode.